DESCRIPTION: West Nile (WN) and Japanese encephalitis (JE) viruses are mosquito-borne flaviviruses that cause a devastating acute neurological illness with up to 30% mortality and permanent neurological disabilities in the survivors. These are also potential category B bioterrorism agents and there is no effective treatment for these viruses. Recently RNA interference, where short RNA molecules (siRNA) mediate the destruction of corresponding mRNA molecules in a sequence-specific manner, has emerged as a tool for potential antiviral therapeutics. After testing several genomic sequences as targets, we have found that a siRNA targeting a highly conserved sequence in the viral envelope gene can protect mice from fatal encephalitis induced by either WNV or JEV. Because many strains of viruses exist in nature that may exhibit small differences even within the conserved sequence, in Aim 1, we will identify an additional 2-3 conserved target sequences that can effectively suppress both JEV and WNV. We will also test if treatment with a combination of siRNAs will increase the magnitude and/or breadth of protection. Additionally to enhance confidence, we will also test the siRNAs for their ability to inhibit multiple viral strains isolated from different geographic locations. In preliminary studies, a single treatment with either lentivirally expressed shRNA or synthetic siRNA was sufficient to provide protection from an infection induced 6 or 18 h earlier. However the currently used method involves administration of siRNA intracranially at the same site as viral challenge and thus, the siRNA is unlikely to diffuse enough to protect all brain cells, which would be required for treatment to be effective after the virus has extensively spread at later time points after infection. Thus in order to realize the siRNA treatment potential in a clinical setting, in Aim 2, we will develop methods for better delivery of siRNA across the brain. Because it will provide the most practical form of treatment, we will develop non- invasive methods for siRNA delivery. To enable uptake by brain cells following intravenous delivery, we will develop pegylated immunoliposomes coated with transferrin receptor antibody as well as use polymeric nanoparticles coated with transferrin. Alternatively, we will try siRNA delivery into the cerebrospinal fluid. If these methods do not work, we will also test delivery efficacy using methods that disrupt the blood-brain barrier transiently. Efficacy will be determined by testing the siRNA's ability to prevent as well as to cure an established infection. [unreadable] [unreadable] [unreadable]